In-vitro amyloid formation occur in both acidic and alkaline condition and there are various methods to detect and quantify the amyloid like Thioflavin-T, Congo red, AFM analysis. Can anybody tell me about pitfall or drawbacks of ThT and Congo red method to avoid false interpretation of data?
For one thing the charge on ThT. We found that it can affect kinetics, even more so at low pH when there are 2 charges on ThT. See the first paper on my profile, on pH dependence of amylin fibrillization.
A brief summary of the problems with ThT can be found in the paper at the bottom. Two main points:
1. In general the intensity response of ThT (and presumably Congo Red) is not linear with the concentration of Abeta in fibrillar form. Large fibers have less surface area than smaller structures with the same cross-beta sheet structure ThT binds to. As a result, you can see the ThT values decrease with time after plateauing due to the creation of mature amyloid fibers from several protofibers.
2. If you are looking at an inhibitor, ThT can competitively bind to the same spot as your inhibitor. This can result in a false positive for inhibition as the inhibitor displaces ThT without destroying the fiber. An example of this is here Biochemistry. Oct 16, 2012; 51(41): 8154–8162. This can be verified more directly by NMR techniques looking at the direct displacement of inhibitors from fibers by ThT (unpublished)
Mr. Jeffrey
So, is there another test that can be replaced by ThT assay or verify the results for the very same reason?
Dear Mona,
congo red assay, nile red ....you can even go for AFM, TEM and CD to visualize amyloid inhibition rather then sticking to the assays.
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